A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum: Production, Purification, and Characterization
| Autor: | Marco A. S. Oliveira, Flavio Augusto Vicente Seixas, Ione Parra Barbosa-Tessmann, Fabiane Cristina dos Santos |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Cellobiose Bioengineering Cellulase 01 natural sciences Applied Microbiology and Biotechnology Biochemistry Sensitivity and Specificity Substrate Specificity Fungal Proteins Hydrolysis chemistry.chemical_compound 010608 biotechnology Enzyme Stability Cellulose 1 4-beta-Cellobiosidase Enzyme kinetics Glycoside hydrolase family 7 Molecular Biology Ammonium sulfate precipitation Phylogeny chemistry.chemical_classification Penicillium digitatum Chromatography biology 010405 organic chemistry Circular Dichroism Penicillium Temperature General Medicine Hydrogen-Ion Concentration biology.organism_classification 0104 chemical sciences Kinetics Enzyme Glucose chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Genome Fungal Biotechnology |
| Zdroj: | Applied biochemistry and biotechnology. 192(1) |
| ISSN: | 1559-0291 |
| Popis: | A new cellulase producer strain of Penicillium digitatum (RV 06) was previously obtained from rotten maize grains. This work aim was to optimize the production and characterize this microorganism produced cellulase. A CMCase maximum production (1.6 U/mL) was obtained in stationary liquid culture, with an initial pH of 5.0, at 25 °C, with 1% lactose as carbon source, and cultured for 5 days. The produced enzyme was purified by ammonium sulfate precipitation and exclusion chromatography. The purified enzyme optimal temperature and pH were 60 °C and 5.2, respectively. The experimental Tm of thermal inactivation was 63.68 °C, and full activity was recovered after incubation of 7 h at 50 °C. The purified 74 kDa CMCase presented KM for CMC of 11.2 mg/mL, Vmax of 0.13 μmol/min, kcat of 52 s−1, and kcat/KM of 4.7 (mg/mL)−1 s−1. The purified enzyme had a high specificity for CMC and p-nitrophenyl cellobioside and released glucose and cellobiose as final products of the CMC hydrolysis. The enzyme trypsin digestion produced peptides whose masses were obtained by MALDI-TOF/TOF mass spectrometry, which was also used to obtain two peptide sequences. These peptide sequences and the mass peak profile retrieved a CBHI within the annotated genome of P. digitatum PD1. Sequence alignments and phylogenetic analysis confirmed this enzyme as a CBHI of the glycoside hydrolase family 7. The P. digitatum PD1 protein in silico structural model revealed a coil and β-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme. |
| Databáze: | OpenAIRE |
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